The present invention is in the field of analyte sensors and, more specifically the present invention is in the field of microcontact printing binders on metal films to produce optical diffraction biosensors.
Microcontact printing is a technique for forming patterns of organic monolayers with micron and submicron lateral dimensions. It offers experimental simplicity and flexibility in forming certain types of patterns. In the prior art, microcontact printing was used with self-assembled monolayers of long-chain alkanethiolates to form organic structures on gold and other metals. These patterns acted as nanometer resists by protecting the supporting metal from corrosion by appropriately formulated etchants, or, allowed for the selective placement of fluids on hydrophilic regions of the pattern. In general, patterns of self-assembled monolayers having dimensions that can be less than 1 micron are formed by using the alkanethiol as an xe2x80x9cinkxe2x80x9d, and by printing them on the metal support using an elastomeric xe2x80x9cstampxe2x80x9d. The stamp is fabricated by molding a silicone elastomer using a master prepared by optical or X-ray microlithography or by other techniques. (See U.S. patent application Ser. Nos. 08/654,993; 08/769,594; 08/821,464; 08/707,456 and 08/768,449 which are incorporated herein in their entirety by reference)
Microcontact printing brings to microfabrication a number of new capabilities. Microcontact printing makes it possible to form patterns that are distinguished only by their constituent functional groups; this capability permits the control of surface properties such as interfacial free energies with great precision. In the prior art microcontact printing relies on molecular self-assembly. Using self-assembling monolayers, a system is generated that is (at least locally) close to a thermodynamic minimum and is intrinsically defect-rejecting and self-healing. Simple procedures, with minimal protection against surface contamination by adsorbed materials or by particles, can lead to surprisingly low levels of defects in the final structures. The procedure using self-assembling monolayers can be conducted at atmospheric pressure, in an unprotected laboratory atmosphere. Thus, microcontact printing that uses self-assembling monolayers is useful in laboratories that do not have routine access to the equipment normally used in microfabrication, or for which the capital cost of equipment is a serious concern. The patterned self-assembled monolayers can be designed to act as resists with a number of wet-chemical etchants.
Also in the prior art, a gold film 5 to 2000 nanometers thick is typically supported on a titanium-primed Si/SiO2 wafer or glass sheet. The titanium serves as an adhesion promoter between gold and the support. However, the silicon wafer is rigid, brittle, and cannot transmit light. These silicon wafers are also not suitable for a large-scale, continuous printing process, such as in letterpress, gravure, offset, and screen printing (see Printing Fundamentals, A. Glassman, Ed. (Tappi Press Atlanta, Ga. 1981); Encyclopedia Britannica, vol. 26, pp. 76-92, 110-111 (Encyclopedia Brittanica, Inc. 1991)). In addition, silicon must be treated in a separate step with an adhesion promoter such as Cr or Ti, or Au will not adequately adhere, preventing formation of a stable and well-ordered monolayer. Finally, silicon is opaque to visible light, so any diffraction pattern obtained must be created with reflected, not transmitted light.
What is needed is an easy, efficient and simple method of contact printing a patterned receptor on an optically transparent, flexible substrate, that is amenable to continuous processing and does not use self-assembling monolayers. Such a method and the device resulting from such a method is simpler, not restricted to the limitations of self-assembling monolayers and is easier to manufacture.
The present invention provides an inexpensive and sensitive device and method for detecting and quantifying analytes present in a medium. The device comprises a metalized film upon which is printed a specific predetermined pattern of analyte-specific receptor. The present invention does not utilize self-assembling monolayers but is more general in that any receptor which can be chemically coupled to a surface can be used. Upon attachment of a target analyte which is capable of scattering light to select areas of the plastic film upon which the receptor is printed, diffraction of transmitted and/or reflected light occurs via the physical dimensions, refractive index and defined, precise placement of the analyte. In the case where an analyte does not scatter visible light because the analyte is too small or does not have an appreciable refractive index difference compared to the surrounding medium, the attachment of polymer beads coupled with the analyte to receptors is another method of producing diffraction of light. A diffraction image is produced which can be easily seen with the eye or, optionally, with a sensing device. The present invention is a biosensor comprising a polymer film coated with metal and a receptor layer printed onto the polymer film wherein the receptor layer has a receptive material thereon that specifically binds an analyte.
The present invention utilizes methods of contact printing of patterned monolayers utilizing derivatives of binders for microorganisms. One example of such a derivative is a thiol. The desired binders can be thiolated antibodies or antibody fragments, proteins, nucleic acids, sugars, carbohydrates, or any other functionality capable of binding an analyte. The derivatives are chemisorbed to metal surfaces such as metalized polymer films.
Patterned monolayers allow for the controlled placement of analytes thereon via the patterns of analyte-specific receptors. The biosensing devices of the present invention produced thereby are used by first exposing the biosensing device to a medium that contains the analyte of choice and then, after an appropriate incubation period, transmitting light, such as from a laser or a point light source, through the film. If the analyte is present in the medium and is bound to the receptors on the patterned monolayer, the light is diffracted in such a way as to produce a visible or near infrared image. In other words, the patterned monolayers with the analyte bound thereto can produce optical diffraction patterns which differ depending on the reaction of the receptors on the monolayer with the analyte of interest. The light can be in the visible spectrum, and be either reflected from the film, or transmitted through it, and the analyte can be any compound or particle reacting with the monolayer. The light can be a white light or monochromatic electromagnetic radiation in preferably the visible region. The present invention also provides a flexible support for a monolayer on gold or other suitable metal or metal alloy.
The present invention includes a support for a thin layer of gold or other suitable material which does not require an adhesion promoter for the formation of a well-ordered monolayer or thin layer of binder. The present invention also provides a support for a layer of gold or other material which is suitable for continuous printing, rather than batch, fabrication. In addition, the present invention provides a low-cost, disposable biosensor which can be mass produced. The biosensors of the present invention can be produced as a single test for detecting an analyte or it can be formatted as a multiple test device. The uses for the biosensors of the present invention include, but are not limited to, detection of chemical or biological contamination in garments, such as diapers, generally the detection of contamination by microorganisms in prepacked foods such as fruit juices or other beverages and the use of the biosensors of the present invention in health diagnostic applications such as diagnostic kits for the detection of antigens, microorganisms, and blood constituents.
In another embodiment of the present invention, nutrients for a specific class of microorganisms can be incorporated into the receptor monolayer. In this way, very low concentrations of microorganisms can be detected by first contacting the biosensor of the present invention with the nutrients incorporated therein and then incubating the biosensor under conditions appropriate for the growth of the bound microorganism. The microorganism is allowed to grow until there are enough organisms to form a diffraction pattern.
The present invention can also be used on contact lenses, eyeglasses, window panes, pharmaceutical vials, solvent containers, water bottles, bandaids, and the like to detect contamination.
These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments.